Human BMP-2 promoter and method for exploring bone-related substance by using the same

ABSTRACT

The present invention provides a method for exploring low molecular weight compounds which regulate positively or negatively the expression of the human BMP-2 with reference to a reporter activity by using 5′ upstream region gene containing the human BMP-2 promoter and an animal cell introduced with a recombinant expression vector which has been connected to an appropriate reporter gene. The low molecular weight susbtances and their derivatives obtained by the present method have morphogenetic activity and inhibiting activity for bone and cartilage through the expression of human BMP-2 and are useful as preventive or therapeutic agents for bone and cartilage diseases.

This application is a 371 of PCT/IB99/00735 filed Apr. 22, 1999.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a 5′ upstream region DNA containing apromoter of a human bone morphogenetic protein (hereinafter calledBMP-2). Further, the present invention provides a method for exploring alow molecular weight compound positively or negatively which regulatesthe expression of human BMP-2 by using a mass of animal cells that areintroduced with a 5′ upstream region DNA containing the human BMP-2promoter and a structure connected to a suitable reporter gene, and byusing a reporter activity as an indicator.

(2) Description of the Related Art

Many substances have been known as usable for healing of bone deficit,fracture, etc. by inducing bone regeneration. Among them, BMP is aprotein belonging to TGF (transforming growth factor) -β superfamily andhas been formerly found as a substance inducing ectopic ossification andexisting in a decalcified product of a bone (Trends in Biotechnol. 11:379-383, 1993). BMP members known so far range from BMP-1 to BMP-14.Among them, the members from BMP-2 to BMP-14 have been known as showingthe bone morphogenetic activity. BMP-2 has been known as having thestrongest activity of bone morphogenesis (Science 242: 1528-1534). Ithas been known that recombinant BMP-2 induces ectopic ossification(Proc. Natnl Acad. Sci. USA 87: 2220-2224, 1990), and is effective tobone deficit in animal model (J. Bone and Joint Surg. 74A, 659-670,1992). The BMP proteins, particularly ranging from BMP-2 to BMP-14, areeffective to therapeutic and preventive treatments of various bonedisturbances and bone diseases. However, the BMP proteins exist in verysmall quantity in nature, and for an available large quantity fortherapeutic use, production of a recombinant protein is necessary. Theproduction of the recombinant protein generally is very expensive ratherthan the preparation of a small molecular substance. Moreover, there aremany restrictions as a medical drug in terms of physical properties oradministration methods due to its proteinic characteristics. Consideringthese points, a low molecular organic compound having the equal activityto that of said BMP protein, if any, will become a highly promisingmedical drug.

For such an exploring method, an example was so far reported using amurine BMP-2 promoter (W097/15308). The region of the murine BMP-2promoter has been already cloned (Biochem. Biophys. Acta, Vol. 1218, p.221-224, 1994), but no report has been published for the region of humanBMP-2 promoter and an exploring method employing the human BMP-2promoter. A comparison of the DNA sequence of 741 bp of the 5′ upstreamexon region of said murine BMP-2 promoter with the corresponding humanBMP-2 promoter sequence (the base sequence No. from 4709 to 5483 shownin SEQ ID NO. 1 of the Sequence Listing) of the present invention shows71.6% homology between these two sequences. Because all the materialsused for establishing the exploring method presented by the presentinvention are of human origin, the substance discovered from explorationcan be quite effective in clinical application.

SUMMARY OF THE INVENTION

The present invention provides a 5′ upstream region DNA containing apromoter of human BMP-2. By using 5′ upstream region gene containing thehuman BMP-2 promoter and an animal cell introduced with the recombinantexpression vector which has been connected to an appropriate reportergene, the low molecular weight compounds which regulate positively ornegatively the expression of human BMP-2 can be explored with referenceto a reporter activity. The low molecular weight compounds and theirderivatives have morphogenetic activity and inhibiting activity for boneand cartilage through the expression of human BMP-2 and are useful aspreventive or therapeutic agents for bone and cartilage diseases,remedies for osteometastasis, or therapeutic and preventive agents forosteohyperplasia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exon-intron structure of 6.7 kb 5′ upstream region of humanBMP-2 gene and a restriction enzyme map. A solid black shows an intronregion.

FIG. 2 is a recombinant expression vector (pBMP2) containing a 5′upstream region of human BMP-2 gene. A SmaI-SacI fragment (base No. from482 to 5345 shown in SEQ ID NO. 1 of the Sequence Listing, referring toFIG. 1) was inserted to the restriction enzyme site of pGL3-basic.

FIG. 3 is a result of measuring a human BMP-2 promoter activity(transiently expression).

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to a DNA containing a human bonemorphogenetic protein-2 promoter region having a base sequence No. from1 to 6728 shown in the SEQ ID NO. 1 of the Sequence Listing, orfragments thereof. The SEQ ID NO. 1 of the Sequence Listing shows the 5′upstream region sequence of the human BMP-2 gene.

The present invention relates to a method for preparing the DNA shown inSEQ ID NO. 1 of the Sequence Listing by conducting the following stepsof:

(1) screening of a P1 phage human genomic DNA library by PCR methodusing a primer represented by SEQ ID NO. 2 and SEQ ID NO. 3 of theSequence Listing,

(2) digestion of the isolated P1 phage with restriction enzymes Sall andSpel,

(3) identification of the aimed fragment by Southern blotting, and

(4) subcloning of the aimed fragment into pbluescript vector cut by thesame enzymes.

The present invention relates to a recombinant expression vectorcharacterized by integration of the DNA shown in SEQ ID NO. 1 of theSequence Listing. The vector integrated is not restricted in particularand can be used among ones commercialized. As a preferable example, pGL3basic vector (a product of Pro Mega Ltd.) was used. It was connected tothe base sequence shown in SEQ No. 1 of the Sequence Listing which hadbeen treated with a restriction enzyme. Then, pBMP2 (9660 bp) wasobtained to be a recombinant expression vector of the present invention.

The present invention relates to a method for exploring a bone-relatedsubstance, characterized by using the full length or a part of DNA shownin SEQ ID NO. 1 of the Sequence Listing that is connected to a reportergene. In other words, the present invention provides the method forexploring a substance that induces BMP-2, which is a member of BMPproteins. Since the substance obtained by the exploring method, althoughit is low molecular weight, has an effect on inducing an expression ofhuman BMP-2, a bone formation factor, it is considered to have an effectequivalent to that of human BMP-2 and to be extremely useful.

In detail, a plasmid vector (above-described recombinant expressionvector) is constructed to locate a suitable region of the 5′ upstreamregion of the human BMP-2 gene shown in SEQ ID NO. 1 of the SequenceListing in front of a reporter gene, and it is necessary to introducethe recombinant expression vector to mammalian cells, preferably humanosteoblast-like cells, such as SaOS-2 cells, with a liposome. The animalcells stably transfected with the recombinant expression vector areselected by using a resistance marker. By such an exploring method,osteogenesis inducing or inhibiting substances can be explored.

The present invention also provides a method for exploring a substance,which inhibits the expression of human BMP-2. If human BMP-2 has somerelationship with bone and cartilage hyperplasia, the hyperplasiadiseases can be prevented by inhibiting the expression.

A low molecular weight compound which induces or inhibits the expressionof human BMP-2 can be obtained by isolating the promoter which regulatesthe expression of the gene, by connecting it to a suitable reporter geneand by introducing the gene structure to a suitable mammal cell to makean exploring system. The reporter gene such as luciferase gene andβ-galactosidase gene shows an expressing status on behalf of an originalproduct. A substance regulating the expression of human BMP-2 in theexploring system works on the promoter to increase or decrease theexpression level of the reporter gene. Therefore, a simple and easymeasurement of the reporter activity makes an exploration of the aimedsubstance possible.

The animal cell transfected with said vector can be used for a methodfor screening a chemical compound library by high throughput screening(Nature, Vol. 384, Suppl., p. 14-16, 1996) and finding an activesubstance from natural substances. The substance which increases ordecreases an activity is searched by treating the cells with a substancefor an appropriate time period and afterwards measuring the reporteractivity. The compound obtained hereby can regulate the expression byworking directly on a transcription factor or indirectly on the promoterof human BMP-2 through regulating a signal transduction system.Therefore, these compounds are effective as a therapeutic agent forosteocartilaginous diseases, cancer metastasis to bone, orosteohyperplasia.

The substance obtained by the present invention has bone or cartilagemorphogenetic activity and is effective as an agent for therapeutic andpreventive treatment in the fields of orthopedic surgery (fracture,osteoarthritis such as joint osteoarthritis and hip jointosteoarthritis, arthrosteitis, damage of cartilage such as damage ofmeniscus, regeneration of bone and cartilage deficit caused by injuryand tumor dissection, bone reconstruction such as spinal fusion andvertebral canal enlargement, and congenital cartilage and bone diseasessuch as dysoteogenesis and achondroplasia), or dental fields (bonereconstruction such as palatoschisis, mandible reconstruction, andresidual ridge construction), and osteoporosis. Moreover, the substanceof the present invention can be used for bone graft in aestheticsurgery. These therapeutic treatments are effective to therapies in thefields of veterinary surgery. On the other hand, the present inventioncan provide a substance which inhibits bone or cartilage morphogenesis.In this case, the substance is applied as an agent for prevention andtherapy of bone and cartilage hyperplasia.

EXAMPLES

This invention shall be more illustratively explained by way of thefollowing Examples.

Example 1

Cloning of Human BMP-2 Genomic DNA

PCR reaction was carried out by using a primer B2-3 shown in SEQ ID NO.2 of the Sequence Listing and a primer B2-7 shown in SEQ ID NO. 3 of theSequence Listing under the condition of 94° C. for 1 min, 60° C. for 30sec, and 72° C. for 30 sec, and a human genomic DNA library of a P1phage vector (a product of Genome System Ltd.) was subjected toscreening. Under this condition, a 270 bp band occurs by using a humanplacenta genomic DNA as a template (a product of CloneTech). P21, one of2 phage vector clones obtained by such method, was digested by SalI andSpeI at 37° C. for 2 hours, respectively. A fragment of ca. 6.7 kblength containing the 5′ upstream region of human BMP-2 gene wasidentified by the conventional Southern blotting method using a primershown in SEQ ID NO. 4 of the Sequence Listing. The fragment of SalI-SpeIwas connected to the pBluescript vector having been treated with thesame enzymes by using Takara Ligation Kit (a product of Takara ShuzoLtd.). The vector was named E. coli P6.7B2 vector. The E. coli P6.7B2was deposited in National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, Ministry of InternationalTrade and Industry 1-3, Higashi 1-chome, Tsukuba-shi Ibaraki-ken305-8566 Japan, in Mar. 30, 1998 with depository number FERM P-16735 andtransferred to the International Depository Authority under BudapestTreaty on Feb. 17, 1999 (Deposit No. FERM BP-6649).

Example 2

Determination of DNA Sequence of Human BMP-2 Promoter

The sequence of the human BMP-2 promoter region was determined by ALFautomatic DNA sequencer (a product of Amersham Pharmacia Biotech Ltd.)using P6.7B2 vector and a vector that was prepared by further subcloningof the inserted fragment of 6.7 kb of said vector as a template. Readingstarted from both ends was repeated at least three times. A sitedifficult in reading was subcloned to read the sequence starting fromboth ends. The sequence of ca. 6.7 kb promoter region and the structurethereof were shown in SEQ ID NO. 1 of the Sequence Listing and FIG. 1,respectively.

Example 3

Construction of a Recombinant Expression Vector that Was Prepared byConnecting a Human BMP-2 Promoter to a Luciferase Reporter Gene

NcoI-SmaI fragment of the 5′ upstream region of the human BMP-2 genecontaining a promoter region was connected to a pGEM5zf (+) vector (aproduct of Pro Mega Ltd.) digested by NcoI and EcoRV. The vectorprepared was treated with NotI and ApaI restriction enzymes andconnected to a pGEM11zf (+) vector (a product of Pro Mega Ltd.) preparedby treatment with the same enzymes. HindIII-SalI fragment of this vectorwas connected to a pGL3-basic vector (a product of Pro Mega Ltd.)treated with HindIII-XhoI. The vector obtained was treated with NcoI andBamHI and connected to a NcoI-BamHI fragment of the BMP-2 gene. Further,the vector obtained was treated with PstI and SacI and connected to aPstI-SacI fragment of the BMP-2 gene to obtain the aimed recombinantexpression vector. The recombinant expression vector obtained was shownin FIG. 2. The recombinant expression vector pBMP2 (9660 bp) containsthe 5′ upstream region of ca. 5.1 kb of the human BMP-2 gene.

Example 4

Measurement of Activity of the Human BMP-2 Promoter

In order to express transiently the recombinant expression vector pBMP2,above-described vectors were mixed with a vector, pRL-SV40 (a product ofPro Mega Ltd.) containing sea pansy luciferase gene as an internalcontrol for measurement of efficiency of gene introduction in an equalquantity. Then, cationic liposome lipofectamine (a product of LifetechOriental Co.) was mixed with thus obtained to add to human osteosarcomacells HOS for transfection. Fire fly luciferase activity and sea pansyluciferase activity were measured by Pikka Gene Dual Kit (a product ofToyo Ink Co.). The result is shown in FIG. 3. The longitudinal axisrepresents a promoter activity that was expressed as a ratio of fire flyluciferase activity to sea pansy luciferase activity. From the result,it has been known that the DNA of SEQ ID NO. 1 of the Sequence Listinghas a promoter activity.

Example 5

Introduction of the Recombinant Expression Vector pBMP2 to a Human Celland Stabilized Expression

In order to express the recombinant expression vector pBMP2 stably, saidvector was mixed with the vector pPUR (a product of CloneTech Ltd.)containing puromycin resistant gene in the proportion of 10:1 and alsomixed with cationic liposome lipofectamine (a product of LifetechOriental Co.) and added to a human osteosarcoma cell HOS fortransfection. The cells to which the aimed gene has been introduced wereselected from a culture medium containing 0.5 μg/mL of puromycin (aproduct of Sigma Ltd.).

Example 6

Screening of an Active Low Molecular Weight Compound

Cells selected were inoculated in a 96-well plate, treated withsubstances of various chemical compound libraries for 1-3 days,dissolved with cytolytic agent (a product of Pro Mega Ltd.), andmeasured for enzyme activity by employing a luciferase assay kit (aproduct of Pro Mega Ltd.). By such processes, various substancesinducing or inhibiting the expression of human BMP-2 can be explored.

4 1 6728 DNA HUMAN misc_feature (1)..(6728) Human BMP-2 5′ upstream genesequence including the intron region. 1 actagtaacc aagtgttact catgctaaaaagccaacagg cttctcaggg gagtcttatt 60 ttgaagacta taatcatttt ccagaagcatgtgctcattc aacccatatt tttgaaatct 120 accaagtgca aaagaaaatc acataagataaactcacatc tttctgagaa ccagaaccga 180 tggccttaat ttgcatgtat atagtaatacaaatttgcaa ccttttgttg tcttcacaca 240 tatcttctca tttattcttc acaacctccatgagaagtca actgttagtc tctatagagt 300 attcatgagg gagccgaggc tatgagaatttgttactgcc atagatcaga ggtccccaac 360 ccccgggcca tgaatagtac ttgtccatggcctgttagga acccccaggg gcaggtgagg 420 gagcattact gcctgagctc tgcctcctgtcagatcagca gcagcattag attctcatag 480 gagctcaaac cctactgtga actgcatgtatgagggatct aggttgcatg ctccttatga 540 gaacctaact aatgcctgac gatctgacgtggaaccgttt catcctgaaa ccatcacccc 600 caacctggtc tgtagaaaaa ttttcctccacaaaactggt ccctggtgcc aaaatggttg 660 gggacctctg ctttagattg actgacccaactggtggcct ttctgatgaa atatccactg 720 tttggcaata taataaaagc tgggagaagagaaggagaag gaggagaaag aggaggagaa 780 gaaaggcatc caaagtccta tttcttaaaaccaggccata ttgcagacag aaggccaaag 840 aatacatttg aagatacata gagtgagtagcaatcagaaa ctaagttcac acatgttatc 900 catttgacct tcatcatgtc agtctgaagcaagcaaacat atatttattt accccaatta 960 acagatgaaa tgaccatttt aaagtcagtcatccatgaca gaaccaggaa ttagaaccca 1020 ggtcacccaa ctacatctta gagtgttatcaacatagttt acagacataa cttaaggtgt 1080 acatcttaaa ctgtgtgaca gtctaatcaatgcacaaaat tcattctttt taatttttaa 1140 gaagatgtag ttggtacttg ctgaaagcagttgatcctga tttggccaac ttggcctagt 1200 ttctgatata aatcactcca cctctcccatcctttctacc atgtctaatc aaacaccaaa 1260 aaggaaaaca tttgattgac taaatgcttctcttttgcct tggctccaag ttcctgtaaa 1320 aaaaacttca tccaggtaaa gtaagtagggatggttatgg gtgtcaccct tgtaacaaag 1380 cgatatgtat gctaaaaagc aaagagggtttctcagggca gttgccacag gtttccatgg 1440 aatgacaatt acaaggtaga tggatcacgtaatgcctgct gtgaacttta caatacatgg 1500 ggatcactat tttaatgttg atttgcataatgtttggcct agcaagaaca atactgaatg 1560 ctaatagcta cagaatgcaa taaaatggaatatcatgtga gctgaagtgc taaaattgct 1620 aaagtatgac tgtaagcagt taatttcatcccgagtcttg tccacacaca atcaaatgca 1680 aatttccact atctcctgag aaactgccaagagatgactg aaatggaata gcaacaacct 1740 cctttatgtg gaatcattat ctggtcatctttgctttttt acataacttg tctgtaccct 1800 ctgatgcatt tgaatttgca acttctactatccctatctt aaacaactcc ccactcccac 1860 ctctgcccct tccacatttg ctggcagatgcactaagtct gataaactca gcaagactga 1920 atctcaccct ctgaactgaa aaactttgaatggacctttg aaaacggtag aattgacaat 1980 ggttagctgc aagtgatatt ttcaaggcaaacagacactc tcccaaagta ttaaataacc 2040 cagcattcta agttgcaggt ggaaggtagccattagtgaa gagagagaaa aaaaaaaaga 2100 aatagctcgt ctgtatttag atttatcatttctgactatt gctcttccct ggaaaacggg 2160 taggtacagt catcctgtac ttcgatcccaaatcagtctc tggagactac ttatttattt 2220 atttatttat ttatggactt ctttctttcaagcgttcgaa ctcatttcca ccacaagagg 2280 gcagccatct ctaaaaaaaa aaaaatagggccaaaattta tgtaagttgt gcttggaaca 2340 agcattcagt agttcctcag aaatcatacaccctacataa aagagattct gcaatgggca 2400 gcactaacat gaaacagtgt tcagaagtacccattttccc tcagattcta aactgacaag 2460 gtttccactt atcaggttat gaagttctaaagctgcaaga catccttgag gtcatcacag 2520 gatatttatt tattttttct tcgggtgcatccaatagtta tcaacttttc ctcctcttta 2580 aaagctactt aaatctcatt gaagttttgttttgttttgt ttttgaaatc taagtaatga 2640 gagaaacaat tgttaacttc tcaattaaacttgataggaa aggaaataat ttcagaagcc 2700 ctgtgtccat gagtaggata tgttttattgcctccttgtt tgcggtgcaa tgactctgag 2760 tgacaatcaa cttctatagc acctttttttttttttttca ggaaataaag tagcatgttc 2820 ctgaataatt cccccacccc cttttattttcctggtagtc aggcttcctc caaaatacct 2880 tatttgacct ttataccttt agaaacagcaagtgcctaat tcgcctctgt gggttgctaa 2940 tccgatttac gtgagcggaa cctagtattattttagctcc cctaccgaaa aaataataca 3000 catggataat agttctatta ccagctcctgcttctgactt ttttctctct gtttcgcagg 3060 cccgatagct ctgggaaagc agaacttggccttttccaaa aattttctgc ccttggtttt 3120 ggggatcatt tgggcaagcc cgaggtgctgtgcatggggg ctcctggaat cctgggaagg 3180 gcagaaagcc ttggccccag actcatcgtgcagcagctct gagcagtatt tcggctgagg 3240 agtgacttca gtgaatattc agctgaggagtgacttggcc acgtgtcaca gccctacttc 3300 ttgggggcct ggtggaagag ggtggcgtagaaggttccaa ggtcccaaac tggaattgtc 3360 ctgtatgctt ggttcacaca gtgcgttattttaccttcct ctgagctgct aatcgcctgc 3420 ctctgagctg ggtgagataa atatcacaaggcacaaagtg attgtacaat aaaaaaatca 3480 aatccctccc atccatcctt cagtctgccacacacgcagt ctacgttaca cacatgtcac 3540 gtaaagcagg atgacatcca tgtcacatacatagacatat tgaccgaaat gtggcccttc 3600 ggttgcatat attctcatac atgaatatatttatagaaat atatgcacat atttttgtat 3660 attggatata tttatgtaac tataaatttacatgcgtatg gatatgaaaa taaatgcata 3720 cacatttatg taaaaaaatt tgtacacatgcatttacata tgtaaataca tacatctcta 3780 tgtattaatg tttaaaaaca ctcaatttccagcctgctgt tttcttttaa ttttcctcct 3840 attccgggga aacagaagcg tggatcccacgtctatgcta tgccaaaata cgctgtaatt 3900 gaggtgtttt gttttgtttt gttttttgaaatcgtatatt accgaaaaac ttcaaactga 3960 aagttgaata acgggcccag cggggaaataagaggccaga ccctgaccct gcatttgtcc 4020 tggatttcgc ctccagagtc cccgcgagggtccggcgcgc cagctgatct ctcctttgag 4080 agcagggagt ggaggcgcga gcgccccccttggcggccgc gcgccccgcc ctccgcccca 4140 ccccgccgcg gctgcccggg cgcgccgtccacacccctgc gcgcagctcc cgcccgctcg 4200 gggatccccg gcgagccgcg ccgcgaagggggaggtgttc ggccgcggcg ggagggagcc 4260 ggcaggggcg tcccctttaa aagccgcgagcgccgcgcca cggcccgccg ccgccgtcgc 4320 cgccgccgga gtcctcgccc cgccgcgctgcgcccggctc gcgctgcgct agtcgctccg 4380 cttcccacac cccgccggga ctggcagccgccgccgcaca tctgccgcca cagcctccgc 4440 cggctacccg aacgttctcg gggccagcgccgagtggatc accggggacc gcgaggcacc 4500 cgcgcgccgc agaccccgcg cgggctggagcacccggcag agcgcgccac agcgccgtgg 4560 cctctgctgc ccggtgcgcc agagccgcggacgggcgcag agcgccgggg actccggagc 4620 cgatccctag cgcgcgatgc ggagcacctactgcaggaga tcgggggcct gggacgcgct 4680 ggccgaggtg tgatcggacc ccaggctagccacaaagggc acttggcccc agggctagga 4740 gagcgagggg agagcacagc cacccgcctcggcggcccgg gactcggctc gactcgccgg 4800 agaatgcgcc cgaggacgac ggggcgccagagcccggtgc tttcaactgg cgagcgcgaa 4860 tgggggtgca ctggagtaag gcagagtgatgcgggggggc aactcgcctg gcaccgagat 4920 cgccgccgtg cccttccctg gacccggcgtcgcccaggat ggctgccccg agccatgggc 4980 cgcggcggag ctaggcggag cgccggaccctcgacccccg agtcccggag ccgccccgcg 5040 cggggccacg cgtccctcgg gcgctggttcctaaggagga cgacagcacc agcttctcct 5100 ttctcccttc ccttccctgc ccggcactcttccccctgct cgctgttgtt gtgtgtcagc 5160 acttggctgg ggacttcttg aacttgcagggagaataact tgcgcacccc actttgcgcc 5220 ggtgcctttg ccccagcgga gcctgttcgccatctccgag ccccaccgcc cctccactcc 5280 tcggccttgc ccgacactga gacgctgttcccagcgtgaa aagagagact gcgcggccgg 5340 cacccgggag aaggaggagg caaagaaaaggaacggacat tcggtccttg cgccaggtcc 5400 tttgaccaga gtttttccat gtggacgctctttcaatgga cgtgtccccg cgtgcttctt 5460 agacggactg cggtctccta aaggtagaggacgcgggcca gggccggggt gggtggtggg 5520 tgggaggggg atttgggcag ccactgcggtagagcccttc cttacgtcca ggccagaagt 5580 aaacagaccc ctctccagtc cacgtgcaaggaggccctgc agggctccca cttccagctg 5640 ccccgggcga ccgtaagcct caccctcccggcccgcactc ttccacccct ctttcttccc 5700 ctctccctgg aatacttttg gagctgttaacacttagatg aggtgtttta tttatttatt 5760 tatttatttt taattttttt aaaaacttttttgggtcaaa gaaatccctt tgagagggta 5820 gcccctggtt tcacccgtta gctgagaacctgtccgctct gccatggtga tctccattct 5880 tcaagtgttt ccgggagact tggtttctttgctcagagcc gtgtcccatt taggaaagta 5940 ctaggagttt ggggttctcc ctacttgtttccagaaatgc gaggggtcag tactgaagga 6000 tcacttggta ctgtgttttt aacagctgacacgtgcatta atagatattc accatttacg 6060 taatcccggg aagatacatg tgtatcttgactgcactgtg gggatgcggg atggagctgc 6120 ctttcgagac acccctgagg gtaggggcctgggacacaag tcataagtgg cttcagaagt 6180 tgtggccttg agcttacagg gtctggaagctataagggtg tgtgtgtgtg tgtgtgtgtg 6240 tgtgtgtgtg tcaggaagtt ctatacagtgcctctaagga agtcacatgc accatttatg 6300 tgtgtttata tgccagacag cgctcagcactccgcatttg ggtttgtata ggggacgcag 6360 ggtgtcagat caagcggtgg ttttcccaggttcccggcat tggctgtcag cgctgtgtca 6420 cacacaaaaa agtgacagtc attggcgctggtttggttgg gggggagggc aaatcccaaa 6480 tctgatgtca gacgagctaa gcgttggatgggagcgataa atcatctggt tcaggaactt 6540 gggacccttc attatcccaa acgtttgagcttcggtcggt cttacctaga ctcgtgagtg 6600 tgccaagcca ggagggcatc ctggaggaggcacgccagcc aaatgggaga ccgggccgcg 6660 ggggcgcgag gggggaggac tgggcggggaactcgggtga ctcacgtcgg tcctgtccgc 6720 aggtcgac 6728 2 23 DNA HUMANmisc_feature (1)..(23) Sense PCR primer B2-3 for screening human BMP-2gene corresponding to the exon of the coding region. 2 gttttgatgtcacccccgct gtg 23 3 23 DNA HUMAN misc_feature Complement((1)..(23))Reverse PCR primer B2-7 for screening human BMP-2 gene corresponding tothe exon of the coding region. 3 cagctggact taaggcgttt ccg 23 4 60 DNAHUMAN misc_feature (1)..(60) Sense PCR 5′-digoxigenin labeled primer forSouthern blotting corresponding to 5′ upstream human BMP-2 gene region.4 ggggacttct tgaacttgca gggagaataa cttgcgcacc ccactttgcg ccggtgcctt 60

What is claimed is:
 1. An isolated DNA whose nucleotide sequencecomprises nucleotides 1 to 6728 shown in SEQ ID No: 1, or a fragmentthereof which is effective as a human bone morphogenetic protein-2promoter.
 2. A recombinant expression vector formed by integrating theDNA of claim 1 connected to a reporter gene into a vector.
 3. A methodof screening for a substance which regulates positively or negativelythe expression of human bone morphogenic protein-2 comprisingintroducing a recombinant expression vector of claim 2 into a mammaliancell and measuring expression of the reporter gene in the presence ofsaid substance to determine the effect of the substance on the humanbone morphogenetic promoter region.
 4. A mammalian cell containing therecombinant expression vector of claim
 2. 5. A screening system fordetecting substances which regulate positively or negatively theexpression of human BMP-2 comprising a cell into which the recombinantexpression vector of claim 2 has been introduced.
 6. The method of claim3, wherein the substance has bone or cartilage morphogenetic activity.7. The method of claim 4, wherein the substance inhibits bone orcartilage morphogenesis.
 8. The screening system of claim 5, wherein thecell is a mammalian cell and the recombinant expression vector isintroduced using liposomes.
 9. The screening system of claim 5, whereinthe cell is a SaOS-2.
 10. The screening system according to claim 5,wherein the recombinant expression vector contains a luciferase orβ-galactosidase reporter gene.
 11. A mammalian cell containing theisolated DNA of claim 1.